We investigate the physiologic regulatory mechanisms for amelogenin expression during enamel formation. In situ hybridization of in vivo developing mouse incisors identified a periodicity for amelogenin transcript accumulation wherein one sagittal row of ameloblasts contained numerous amelogenin transcripts while the adjacent parallel rows of ameloblasts showed progressively diminishing numbers of amelogenin transcripts. Ameloblast-like cell line LS8 shows a circadian cycle for amelogenin transcript abundance that cycles out of phase to the negative limb of the circadian rhythm. By binary gene ablation, we knocked-out C/EBP-alpha expression in ameloblasts and found that C/EBP-delta compensated to maintain amelogenin mRNA abundance. In LS8 cells we showed that C/EBP family members pair with NF-Y to synergistically activate the amelogenin promoter. We identified transcription factors YY1 and Msx2 as opposing amelogenin transactivation. C/EBP-alpha over expression in LS8 cells increases the expression of the alternatively spliced mouse 59 amino acid (M59) long amelogenin mRNA isoforms. M59 protein shows limited ability to self-assemble into protein nanospheres while growing evidence suggests that M59 can provide physiologically relevant signals to ameloblasts. We hypothesize that amelogenin mRNA periodicity and isoform abundance is a consequence of transcription factor interactions on the promoter. The physiologic role for periodicity in amelogenin abundance is to coordinate matrix production with mineral replacement. We hypothesize that linkage between the ameloblasts with the protein matrix participates in providing feedback to ameloblasts serving to coordinate transcription, including to stop amelogenin expression at a predetermined enamel thickness. To test these hypotheses we propose four interrelated specific aims. Specific aim 1: Use chromatin immunoprecipitation (ChIP) strategies to determine the contribution of C/EBP-alpha, C/EBP-delta, NF-Y, YY1 and Msx2 to regulating the amelogenin promoter. Specific aim 2: Determine the contribution of C/EBP-alpha and C/EBP-delta to generate amelogenin mRNA isoforms. Specific aim 3: Determine the role of selected amelogenin isoforms and amelogenin degradation fragments to provide feedback signals to ameloblasts altering transcript abundance and stability. Specific Aim 4: Examine the role of circadian transcription factors to regulate amelogenin transcript abundance. By exploring the physiologic pathways responsible for regulating the unique amelogenin gene, these studies will provide a better understanding of tooth development while contributing to our general understanding of gene regulation. Knowledge of these pathways will contribute to enamel regeneration strategies. Public Health Relevance: Enamel caries is the most prevalent infectious disease of man. Correct orchestration of enamel gene expression is essential to the formation of the enamel organic matrix that must be created first in order to guide enamel biomineralization. We are studying the transcription factors responsible for expression of the dominant protein of forming mammalian enamel, amelogenin, and the circadian rhythms that influence cyclic oscillation in the abundance of amelogenin transcripts.